On the mode of action of edeine

On the mode of action of edeine

578 BIOCHIMICA ET BIOPHYSICA ACTA BBA 95196 ON THE I. EFFECT MODE OF ACTION OF EDEINE IN A CELL-FREE ON OF THE EDEINE SYNTHESIS OF POLYP...

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578

BIOCHIMICA ET BIOPHYSICA ACTA

BBA 95196

ON

THE

I. EFFECT

MODE

OF

ACTION

OF EDEINE

IN A CELL-FREE

ON

OF THE

EDEINE SYNTHESIS

OF POLYPHENYLALANINE

SYSTEM

M. H I E R O W S K I * ANn Z. K U R Y L O - B O R O W S K A The Rockeleller Institute, New York, N.Y. (U.S.A.) (Received August 28th, 1964)

SUMMARY

Edeine in concentrations as low as IO-v M can inhibit the synthesis of polyphenylalanine in a cell-free system. Amino acid incorporation stimulated b y synthetic polynucleotides is inhibited stronger than the incorporation stimulated by added natural templates (Escherichia coli RNA or RNA of ZINDER'S coliphage f2), whereas endogenous incorporation is only slightly inhibited. Edeine has no effect on the transfer of amino acids to the soluble (or transfer) RNA but strongly inhibits the polymerization of phenylalanine from aminoacyl soluble RNA on ribosomes, by inhibiting the binding of aminoacyl soluble RNA to ribosomes. Edeine does not affect the binding of polyribouridylic acid to ribosomes. When the ternary complex of ribosome, polyribouridylic acid and phenylalanyl soluble RNA is formed prior to the addition of edeine, this drug has no effect on the polymerization of the amino acids.

INTRODUCTION

Edeine is a polypeptide antibiotic which inhibits the synthesis in vivo of bacterial DNA, not affecting the synthesis of RNA 1-3. Although the inhibition in vivo of protein synthesis did not appear to be as significant as the strong inhibition of DNA synthesis, the data presented in this paper show that this antibiotic can inhibit the polynucleotide-stimulated incorporation of amino acids by interfering with the attachment of aminoacyl s-RNA to the ribosomes during the course of a polynucleotide-dependent reaction. Abbreviations: Poly-A, polyriboadenylic acid; poly-C, polyribocytidylic acid; poly-U, polyribouridylic acid. Poly-UA, poly-UC and poly-UG are the corresponding copolymers. s-RNA, soluble or transfer ribonucleic acid; m-RNA, messenger RNA; f2 phage RNA, ribonucleic acid of Zinder's coli phage f2. * Present address: D e p a r t m e n t of Biochemistry, Academy of Medicine, Poznan, Poland.

Biochim. Biophys. Acta, 95 (1965) 578-589

ON THE MODE OF ACTION OF EDEINE

579

MATERIAL AND METHODS Crude extract o/ Escherichia coli B Frozen cells were ground with 2.5 times their weight of alumina and extracted with 3 volumes of 5 mM Tris buffer (pH 7.4), containing IO mM MgC12 and 5.0 ~g of deoxyribonuclease (EC 3.1.4.5) per ml. All manipulations were carried out at 4 °. The crude extract was centrifuged at 3o ooo × g for 30 rain.

Assay system This was prepared by the method of NIRENBERG et al. 4 as follows: 3o ooo ×g supernatant was divided into two parts. One part was dialysed against IOO volumes of 50 mM Tris buffer (pH 7.4) with IO mM MgCI,, 60 mM KC1 and 6 mM GSH. This fraction was designated as the "30 o o o × g non-preincubated supernatant". The second portion of the supernatant was incubated at 35 ° for 30 min with the components required for the amino acid incorporation into protein: 3 mM ATP, 0.2 mM GTP, IO mM phosphoenolpyruvate, IO mM GSH, 3o.o#g of pyruvate kinase (EC 2.7.1.4o) per ml, IO mM MgC12, 30 mM KC1, 50 mM Tris buffer (pH 7.8), 4 o # M of each amino acid, 5.0 #g of deoxyribonuclease per ml and I.O mg of s-RNA per ml. After incubation the mixture was dialysed overnight against IOO volumes of o.Io M Tris buffer (pH 7.8) in IO mM MgC12, 30 mM KC1 and 5 mM GSH. This mixture was designated as the "30 ooo ×g preincubated supernatant" fraction. All supernatant fractions were stored at --20 ° .

Isolation o/ribosomes Ribosomes were isolated from the deoxyribonuclease-treated 3o o o o × g supernatant fraction by 3 h centrifugation at lO5 ooo ×g. The ribosomal pellet was washed 3 times b y repeated centrifugation and resuspension in IO mM Tris buffer (pH 7.4) with IO mM MgC12. The ribosomes were resuspended in the same buffer and the suspension was stored frozen.

R N A preparations E. coli R N A was prepared according to the method of NIRENBERG AND MATTHAEI4. E. coli s-RNA was prepared after the method of YON EHRENSTEIN AND LIPMANNs. The f2 phage RNA was generously provided b y Dr. N. ZINDER.

Other materials The ternary complex of ribosomes-poly-U-phenylalanyl s-RNA was prepared after NAKAMOTO~. Poly-[I*CIU was prepared by the method of CHAPEVILLE et al. 7 using the enzyme polynucleotide phosphorylase (EC 2.7.7.8) and [14ClUDP. The specific activity of the preparation was 6.35-1o 4 counts/min per mg. Polynncleotide phosphorylase was obtained b y the method of SINGER et al. 8. The enzyme polymerizing phenylalanine to polyphenylalanine was prepared from E. coli B by the method of CONWAY9. Edeine was prepared as described before lo. Synthetic l?iochim. Biophys. Acta, 95 (1965) 578-589

580

M. HIEROWSKI, Z. KUI{YLO-B(IRtVWSt<:\

polynucleotides: poly-A, poly-U, poly-C, poly-UA, poly-UC and poly-UG were the products of Miles Company, Clifton, New Jersey. Sodium salts of ATP and GTP were obtained from Pabst Laboratories. The trisodium salt of phosphoenolpyruvate and pyruvate kinase were products of Boehringer Company, Mannheim, Germany. [14C~UDP and [14Clamino acids were purchased from Schwarz Bioreseareh Inc., Mt. Vernon, N.Y. Deoxyribonuclease (2 times recrystallyzed) was a product of Worthington Biochemical Corporation.

The basic reaction mixture The basic reaction mixture contained in the total volume of o.25 ml following components: 25.o#moles Tris buffer (pH 7.8), 2.5/*moles MgC12, I2.5 #moles KCI, 1.5/,moles fl-mercaptoethanol, 0.25/,moles disodium salt of ATP, 0.o25/*moles disodium salt of GTP, 1.25/*moles phosphoenolpyruvate, 5.0 #g pyruvate kinase.

Radioactivity measurement The radioactivity of experimental samples was measured in the 5 % trichloroacetic acid precipitates, hot or cold, as indicated for each experiment. The precipitates were washed four times with the same acid and were collected on a Millipore filter. After drying the radioactivity was measured in a windowless gas flow counter for IO rain.

RESULTS

Effect o/ edeine on the synthesis on poly[14Clphenylalanine The effect of edeine on the poly-U-stimulated incorporation of [14C~phenylalanine into acid-insoluble poly[a4C~phenylalanine was studied in the system described by NIRENBERG AND MATTHAEI4. The data presented in Fig. IA show that edeine inhibits the incorporation of [14C]phenylalanine into poly[14Clphenylalanine at a minimal concentration of 0.06 #M. Maximum of inhibition occurs at a 0.5 uM concentration.

E[[ect o/ Mg 2+ concentration on the inhibitory action o/edeine To assess the role of Mg~+ in the inhibitory action of edeine, the poly-Udependent incorporation of [14Clphenylalanine into poly [xaC]phenylalanine was carried out in the presence of two concentrations of edeine: 0.25 #M and 0.5/*M and various concentrations of MgClz. Fig. 2 shows that 0.25 #M edeine induces 50 % inhibition of [14C]phenylalanine incorporation as compared with the control. This ratio is constant in the presence oi o.64-1.85 mM of MgC12. At the same concentrations of MgC12 0.5 #M edeine causes 94-98 % inhibition of [14Clphenylatanine incorporation. The data in Fig. 2 show that the Mg 2+ concentration has no significant effect on the inhibitory action of edeine. Edeine does not displace an optimum of Mg ~+ for poly-U-dependent [14C~phenylalanine incorporation to the higher concentration as is true for streptomycin n. Biochim. Biophys. Acta, 95 (1965) 578-589

ON THE MODE OF ACTION OF EDE1NE

5~I

B

_~ooo 100

c E

x-~-//.----×

g F

IOO0

~6 50

:~_

8

I .50 0 o o 25 Concentration of Edeine ( I J M )

o65

:}Ede,

OS M

.

10

15

20

5

8_ "o

Minutes

Fig. IA. E f f e c t of edeine c o n c e n t r a t i o n on t h e i n c o r p o r a t i o n of [HC]phenylalanine into polyp h e n y l a l a n i n e , o.25-ml s a m p l e s c o n t a i n e d 3 ° ooo x g p r e i n c u b a t e d s u p e r n a t a n t fraction of E. coli ]3 e q u a l to 1.2 m g of protein, 5.0 # g p o l y - U in t h e b a s i c r e a c t i o n m i x t u r e , 0.25 m g s - R N A , 5.0 m/~moles [a4C]phenylalanine w i t h 33 ooo c o u n t s / m i n (specific a c t i v i t y 141 ffC//~mole) a n d 25.0 # m o l e s of e a c h a m i n o acid e x c e p t p h e n y l a l a n i n e . S a m p l e s were i n c u b a t e d for 3 ° m i n a t 35 ° in t h e p r e s e n c e of edeine in c o n c e n t r a t i o n s as i n d i c a t e d in t h i s figure. R a d i o a c t i v i t y w a s m e a s u r e d in h o t 5 ~o trichloroacetic acid p r e c i p i t a t e s . Fig. IB. I n h i b i t i o n of p o l y - U - d e p e n d e n t i n c o r p o r a t i o n of [14C]phenylalanine as a f u n c t i o n of t i m e of t h e a d d i t i o n of edeine. T h e r e a c t i o n m i x t u r e was p r e p a r e d as described for F i g u r e IA. E d e i n e w a s a d d e d to t h e final c o n c e n t r a t i o n of 0. 5 ffM a t zero t i m e of i n c u b a t i o n or 5 rain after t h e o n s e t of i n c u b a t i o n . I n 5 - m i n i n t e r v a l s s a m p l e s i n c u b a t e d a t 35 ° were w i t h d r a w n a n d p r e c i p i t a t e d w i t h h o t 5 % trichtoroacetic acid. R a d i o a c t i v i t y of p r e c i p i t a t e s w a s m e a s u r e d as described in METHODS. A

~

/ /

3oo(

E E

o Control

\ \

Edeine [] 0 . 2 5 p M M

2ooc

c

>i u o 1000 £ 13

0

0.64

1,28

1.92

C o n c e n t r a t i o n of Mg 2. ions (~JM)

Fig. 2. E f f e c t of Mg ~+ c o n c e n t r a t i o n on t h e p o l y - U - d e p e n d e n t i n c o r p o r a t i o n of [a4C]phenylalanine in t h e p r e s e n c e of edeine. T h e r e a c t i o n m i x t u r e w a s p r e p a r e d a n d i n c u b a t e d as described for Fig. IA. o.25-ml s a m p l e s were i n c u b a t e d in t h e presence of MgC12 as i n d i c a t e d in t h i s figure. R a d i o a c t i v i t y w a s m e a s u r e d in h o t 5 °/o trichloroacetic acid precipitates. O, control; F], w i t h 0.25 ffM edeine; A, w i t h o.5o/*M edeine.

Biochim. Biophys. Acta, 95 (1965) 578-589

582

M. HIEROWSK1, Z. KURYLO-BOI{OWSKA

Effect o/ the time o~ edeine addition on incorporation o~ [t4Clphenylalanine In the experiments carried out in the presence of o.5 ffM edeine added at zero time and 5 rain after the onset of incubation of the reaction mixture it was found that the time of edeine addition is important. Fig. I B illustrates the results obtained in the presence of o.5 ffM edeine added simultaneously with the other components of the reaction mixture, and at 5 rain after the onset of incubation. Edeine added 5 rain after the onset of incubation has no effect on poly-U-dependent incorporation of [14Clphenylalanine, whereas added at zero time induces about 94 o/ /o inhibition. Presumably this inhibition occurs only if edeine is added before the formation of a functional ternary complex of phenyla!anyl s-RNA, ribosomes and poly-U. The results of detailed studies of this phenomenon will be discussed later.

Effect o~ edeine on the incorporation o/amino acids stimulated by various polynucleotides The effect of edeine on amino acid incorporation stimulated b y synthetic homopolymers, copolymers and various ~RNA's obtained from natural sources was studied. The experiments carried out with endogenous m - R N A of E. coli show, that if the m - R N A is naturally attached to ribosomes the incorporation of [x4C]phenylalanine into protein is resistant to the action of edeine (Fig. 3A). Some inhibition oc-

~oc

's{

5c

O ' 4

'Q25

0'5

"/--"-5,0 025 05 / A - - 5.0 0.25 0..5 Concentr'mion of Edeine (HM)

~

510

Fig. 3. E f f e c t of edeine on p o l y - U - d e p e n d e n t a n d E. coli R N A - d e p e n d e n t i n c o r p o r a t i o n of [14C]p h e n y l a l a n i n e into p o l y p h e n y l a l a n i n e , o.25-ml s a m p l e s c o n t a i n e d t h e basic r e a c t i o n m i x t u r e , o . o i / * m o l e of e a c h a m i n o acid e x c e p t p h e n y l a l a n i n e , o.oo 5 / , m o l e s [l*C~phenylalanine (specific a c t i v i t y i o / , C / / , m o l e ) a n d : (A) 30 o o o × g n o n - p r e i n c u b a t e d s u p e r n a t a n t fraction of E. coli B e q u a l to 1.2 m g of protein. (B) 3 ° ooo × g p r e i n c u b a t e d s u p e r n a t a n t fraction (the s a m e a m o u n t as above) w i t h o.25 m g of E. coli B R N A ( × ). S a m p l e s w i t h o u t E. coli R N A were u s e d as a control for t h i s e x p e r i m e n t ( O ) . (C) 3 ° ooo × g p r e i n c u b a t e d s u p e r n a t a n t fraction of E. coli B w i t h 5 . 0 / , g of poly-U. All s a m p l e s were i n c u b a t e d for 3 ° rain a t 35 ° in t h e presence of edeine in t h e conc e n t r a t i o n as i n d i c a t e d in t h e figure above. R a d i o a c t i v i t y w a s m e a s u r e d in h o t trichloroacetic acid p r e c i p i t a t e s .

curs (about 35 %) when edeine and E. coli R N A are added to the incubation mixture which contains ribosomes deprived of their natural messenger (Fig. 3B). Greater inhibition occurs if f2 phage RNA is the source of m - R N A (Table I). The poly-Udependent incorporation of [14C]phenylalanine is very sensitive to edeine (Fig. 3C) in contrast to chloramphenicoP ~. At a 0.5 #M concentration of edeine the incorporation of [14Clpbenylalanine stimulated b y poly-UG, poly-UC, or poly-UA is also strongly Biochim. Biophys. Acta, 95 (1965) 578-589

ON THE MODE OF ACTION OF EDEINE TABLE

583

I

I~FFECT O F E D E I N ~

ON THE

f2 P H A G E

RNA-sTIMULATED

INCORPORATION

OF

[liCIPHENYLALANINE o.25-ml s a m p l e s contained 3 ° ooo x g p r e i n c u b a t e d s u p e r n a t a n t fraction of E. coli B equal to 1.2 mg of p r o t e i n in t h e basic reaction m i x t u r e , o.oI jumole of each a m i n o acid except phenylalanine, o.oo5 #moles [l~C]phenylalanine (specific activity lO.o~uC/~umole) and 5o.o~ug of f2 p h a g e RNA. Samples were i n c u b a t e d for i o m i n at 35 ° w i t h o u t or w i t h o. 5 #M and 5.o/~M edeine. R a d i o a c t i v i t y of h o t 5 % trichloroacetic acid precipitate was m e a s u r e d as described in ~ETttODS.

/2 phage R N A (#g)

Conch. o/ edeine (l~M)

[14C]Phenylalanine (#*l~moles)

Incorporation (% inhibition)

o o o 5o.o 50.0 50.0

o.o o. 5 5.o o.o o.5 5 .0

3o.o 19.o 18.o 152.o 65.o 59.3

-36.o 4o.o -57.0 61.o

inhibited (about 95 %). With o.25 #M edeine differences in the per cent of inhibition are observed with these copolymers. These differences can be accounted for by the uridylic acid content of the copolymers. The per cent of inhibition increases with the increase of uridylic acid in the copolymer (Fig. 4A). In the presence of 0.25 #M edeine and homopolymers such as poly-A, poly-C and poly-U, the poly-A-dependent incorporation of [14C]lysine is more inhibited than the poly-C-dependent incorporation of [14Clproline, or the poly-U-dependent incorporation of [l~C]-phenylalanine (Fig. 4B). In the presence of 0.5 #M edeine 95 ~o inhibition of the incorporation of the above amino acids is observed.

E[[ect o/ edeine on the trans/er o/ [14C]phenylalanine to s-RNA It was shown previously that the time of addition of edeine to the reaction mixture is very important in the inhibition of poly-U-dependent incorporation of [14C3phenylalanine into polyphenylalanine (Fig. IB). Further studies were undertaken to determine the step in the synthesis of polyphenylalanine which is inhibited by edeine. The results presented in Table II show that 0.5 #M and 5.o #M edeine have no effect on the transfer of [laClphenylalanine to the s-RNA even though 0.5 #M is sufficient to give complete inhibition of poly-U-stimulated [~4C]phenylalanine incorporation.

E]]ect o/ edeine on polyphenylalanine synthesis /rom [14Clphenylalanyl s-RNA In order to determine whether or not edeine affects the energy-dependent polymerization of [14C]phenylalanyl s-RNA the following procedure was used. (I). Edeine was added to the reaction mixture containing E14C]phenylalanyl s-RNA, poly-U, ribosomes, GTP, and polymerizing enzymes 5 min after the onset of incubation. (2). Edeine was added at zero time. (3). The control samples were incubated in the absence of edeine. All samples were incubated for 7 min. The results presented in Fig. 5 show that edeine exerts a strong effect on the energy-dependent formation of polyI14C~phenylalanine from [14Clphenylalanyl s-:RNA, o.5#M concentration of edeine added to the reaction mixture at zero time causes a 9 ° % inhibition of the Biochim. Biophys. Acta, 95 (1965) 578-589

584

M. HIEROWSKI, Z. KURYLO-BORO~,VSKA A

ec "E _a a c~ 100

• Poly -U

A Poly -UC a Poly-UC x Poly- UC o Poly-UA

t ~ / 5 I oa ~100t,

1:1 8:1 5:1 (5:1

B ~ P o l y - C - pr, oline • Poly - U - phenylafonine x Poly-A-ly,~ine

¢x

%"

o

50

o

z

025

50

050

025

050



Edeine concentration(lJM) Fig. 4. E f f e c t of edeine on t h e s y n t h e t i c p o l y n u c l e o t i d e s - s t i m u l a t e d i n c o r p o r a t i o n of a m i n o acids. A. I n h i b i t i o n b y edeine of t h e i n c o r p o r a t i o n of [14C]phenytalanine s t i m u l a t e d b y poly-UC, p o l y - U G a n d p o l y - U A , o.25-ml s a m p l e s of t h e basic r e a c t i o n m i x t u r e w i t h 5.o # g of s y n t h e t i c p o l y n u c l e o t i d e were i n c u b a t e d for 3 ° m i n a t 35 ° in t h e presence of edeine in t h e c o n c e n t r a t i o n s as i n d i c a t e d in t h i s figure. R a d i o a c t i v i t y of h o t 5 % trichloroacetic acid p r e c i p i t a t e s w a s m e a s u r e d as d e s c r i b e d in METHODS. C o r r e s p o n d i n g b l a n k v a l u e s o b t a i n e d for t h e s a m p l e s w i t h t h e s a m e c o n c e n t r a t i o n of edeine b u t in t h e a b s e n c e of p o l y n u c l e o t i d e s were s u b s t r a c t e d f r o m each exp e r i m e n t a l v a l u e a n d t h e i n c o r p o r a t i o n was e x p r e s s e d as a p e r c e n t of t h e i n c o r p o r a t i o n of t h e n o n - i n h i b i t e d control, ioo % of i n c o r p o r a t i o n w a s r e p r e s e n t e d b y : 1.46 r e # m o l e s of It4C]phenyl a l a n i n e i n c o r p o r a t e d in t h e presence of p o l y - U C (8 : i ), 0.23 m # m o l e s i n c o r p o r a t e d in t h e presence of p o l y - U C (i : i), 0. 5 r e # m o l e s i n c o r p o r a t e d in t h e presence of p o l y - U A (6 : I ) a n d 0.59 r e # m o l e s i n c o r p o r a t e d in t h e p r e s e n c e of p o l y - U G (5:1). B. I n h i b i t i o n b y edeine of i n c o r p o r a t i o n of a m i n o acids: L-[~C~proline, L-[UCllysine a n d L-I~4C] p h e n y l a l a n i n e s t i m u l a t e d b y poly-C, p o l y - A a n d poly-U. I n c o r p o r a t i o n of t-[14Clproline, L[l*C]lysine or L-[l*C]phenylalanine w a s m e a s u r e d in t h e presencc of: i o o / , g poly-C, 5 # g poty-A or 5 # g p o l y - U respectively. E d e i n e was a d d e d s i m u l t a n e o u s l y w i t h t h e h o m o p o l y m e r s in t h e c o n c e n t r a t i o n as i n d i c a t e d in t h i s figure. T h e a s s a y c o n d i t i o n s were t h e s a m e as for Fig. i. To t h e i n c u b a t i o n m i x t u r e w a s a d d e d o n l y this a m i n o acid (5.o r e # m o l e s w i t h a specific a c t i v i t y IO pC/ # m o l e ) w h o s e i n c o r p o r a t i o n w a s m e a s u r e d . T h e p r e c i p i t a t i o n of L-[liC~proline w a s carried o u t w i t h 20 % trichloroacetic acid a f t e r a d d i t i o n of i o o # g of poly-L-proline as a carrier. T h e prec i p i t a t i o n of poly-L-[t4Cllysine a n d p o l y - [ t 4 C l p h e n y l a l a n i n e was carried o u t as described b y GARDNER et al. is. P r e c i p i t a t e s r a d i o a c t i v i t y w a s m e a s u r e d as described in METHODS. C o r r e s p o n d i n g b l a n k v a l u e s o b t a i n e d in t h e s a m e w a y as described for Fig. 4 A were s u b t r a c t e d f r o m t h e experi m e n t a l points, a n d t h e i n c o r p o r a t i o n w a s e x p r e s s e d as a per c e n t of t h e n o n - i n h i b i t e d control. I O 0 o/ / o of i n c o r p o r a t i o n w a s r e p r e s e n t e d by: 1.22 i n # m o l e s of [14C]phenylalanine, 0.21 r e # m o l e s of [14C]lysine a n d o.i 3 m # m o l e s of E14C~proline. TABLE

II

O N T H E T R A N S F E R O F [ 1 4 C ] P H E N Y L A L A N I N E TO s - R N A o.25-ml s a m p l e s c o n t a i n e d 3 ° ooo × g s u p e r n a t a n t f r a c t i o n of E. coli B e q u a l to 1.2 m g of protein, 25.o # m o l e s Tris b u f f e r (pH 7.4), 2.5 # m o l e s MgC12, I.O # m o l e f l - m e r c a p t o e t h a n o l , 0. 5 # m o l e s [14C]phenylalanine (specific a c t i v i t y i41 # C / # m o l e ) a n d o.25 m g s - R N A . S a m p l e s were i n c u b a t e d w i t h o u t or w i t h o.5 # M a n d 5.o # M edeine for io m i n at 35 °. R a d i o a c t i v i t y w a s m e a s u r e d in cold 5 % trichloroacetie acid p r e c i p i t a t e s . EFFECT

OF E D E I N E

Concn. of edeine (#M)

[14C] Phenylalanine trans[erred to s - R N A (counts/min per rng)

o 0.5o 5.oo

4392 4448 4420

Biochim. Biophys. Acta, 95 (1965) 578-589

ON THE MODE OF ACTION OF EDEINE

585

4OO

x

~'EcC300200 .>

z ~_

100

I

I

I

I

I

I

Timeof incubation(rain) 1

3

5

I

7

Fig. 5- Effect of edeine on t h e transfer of [14C]phenylalanine from [14C]phenylalanyl s - R N A t o t h e ribosomal protein, o.25-ml samples contained: 12. 5/zmoles Tris buffer (pH 7.8), 2. 5/~moles MgCI,, 1.5#moles fl-mercaptoethanol, 4o.o/zmoles NH4C1, 0. 5 mg ribosomes, 5.o/,g poly-U and o.03 m g [l*C]phenylalanyl s - R N A (specific activity 141/zC//~mole). After 5 min i n c u b a t i o n a t 35 ° 5o.o/zg of polymerizing e n z y m e s and o.25/*moles of G T P were a d d e d and i n c u b a t i o n c o n t i n u e d for 7 min w i t h o u t ( × ) or w i t h 5.o/zM edeine ( A ) . Simultaneously samples w i t h 5.o/~M edeine were i n c u b a t e d for 5 min prior to t h e a d d i t i o n of polymerizing e n z y m e s (O). The radioa c t i v i t y was measured in h o t trichloroacetic acid precipitates as described in ~aETHOOS. A

B

Counts Absorboncy ot 260rap

:L

E

E

10c

2.0"

O

I00 2

w

o

v

"~ 5C

?, o

8

50 "7

1.0"

/

8 o 0L

5 iO

---r----

10 Tube number

i

20

i

30

i

40

5'0

Fig. 6. E f f e c t of edeine on t h e f o r m a t i o n of t h e t e r n a r y complex, o.25-ml samples containing 12.5 # m o l e s Tris buffer (pH 7.8), 3.o/,moles MgC1 v 4 ° / z m o l e s NH4C1 , o.8 m g of ribosomes, 5.o/*g poly-U and 0.08 mg of [14C]phenylalanyl s - R N A (specific a c t i v i t y 297/,C//,mole) were inc u b a t e d for IO m i n a t 25 ° w i t h o u t (A) or w i t h (B) I.O #,IV[ edeine. After i n c u b a t i o n 0.2 ml of each s a m p l e were centrifuged in t h e exponential d e n s i t y g r a d i e n t of sucrose (5-22 %) containing o.o 5 M Tris buffer (pH 7.4), o.12 M MgC12 a n d o:16 M IqH4C1. Samples were centrifuged in a Spinco SW-39 r o t o r at 4 ° for 6o m i n at 39 ooo rev./min. The r a d i o a c t i v i t y ( x ) and t h e a b s o r b a n c y at 260 m/, (©) were m e a s u r e d in t h e 2-drop fractions collected from each sample after t h e centrifugation.

Biochim. Biophys. Acta, 95 (1965) 578-789

586

M. HIEROWSKI, Z. KURYLO-BOROWSKA

transfer of [laC~phenylalanyl s-RNA to polyphenylalanine. The same amount of edeine added 5 rain later has no effect on this transfer. The data show, therefore that polymerization is not inhibited if ribosomes, phenylalanyl s-RNA and poly-U are preincubated to form the functional ternary complex of these nucleic acids ~3-~5. E//ect o/ edeine on the binding o~ aminoacyl s - R N A to ribosomes NAKAMOTO et al. 18 and SPYRIDES15 have reported that the formation of the ternary complex between ribosomes, poly-U and aminoacyl s-RNA is not energy dependent, and that the formation of this complex is not affected b y chloramphenicol and puromycin. In order to determine whether edeine has an effect on s-RNA binding to ribosomes the following experiments were carried out. The reaction mixtures containing poly-U, ribosomes and s-~RNA charged with [14Clphenylalanine were incubated for IO rain at 25 ° in the presence of 5.0 #M edeine. A sample incubated without edeine was used as a control. After incubation the samples were layered on top of a logarithmic sucrose-density gradient (5-22 %) and centrifuged for 60 rain at 39 ooo rev./min. After centrifngation 2-drop fractions were collected. The radioactivity and the absorbancy at 260 m# of the fractions were measured. Fig. 6 shows the sedimentation pattern ol~*ained in the absence (Fig. 6A) and in the presence (Fig. 6B) of edeine. The curve of the radioactivity obtained for the sample incubated with edeine shows that the binding of aminoacyl s-RNA to ribosomes is inhibited b y this drug. Et/ect o/ edeine on binding o/poly-[l*C]U to ribosomes The effect of edeine on the binding of poly-[14C~U to ribosomes was studied by means of sucrose density-gradient analysis. Fig. 7 illustrates the results of an exA x--×

Counts

Absorboncy at 2 6 0 mp

E

~-100

2,0-

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-o

E

g o

5o

1.0-

~ 5c o

o o r~

c~

10

20

30

nn nn

J

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I

20

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30

Fig. 7. E f f e c t of edeine o n t h e f o r m a t i o n of t h e c o m p l e x b e t w e e n r i b o s o m e s a n d poly-[14C]U. o.25-mi s a m p l e s c o n t a i n i n g Tris buffer, MgCI, a n d N H , C I in t h e s a m e a m o u n t s as described for Fig. 6 were i n c u b a t e d for io m i n a t 25 ° w i t h o.8 m g of ribosomes, 3 . o / , g poly-[laCJU w i t h o u t (A) edeine or w i t h (B) i . o / , M edeine. A f t e r i n c u b a t i o n o.2 ml of e a c h s a m p l e s were centriffuged in t h e d e n s i t y g r a d i e n t of s u c r o s e in t h e s a m e w a y as described for Fig. 6. T h e r a d i o a c t i v i t y ( × ) a n d a b s o r b a n c y a t 260 m # ( © ) were m e a s u r e d as before.

Biochim. Biophys. Acta, (1965) 95 578-589

687

ON THE MODE OF ACTION OF E D E I N E

periment in which poly-[~4ClU was incubated with ribosomes in a reaction mixture without and with I.O #M edeine. Samples were incubated for IO min at 25 ° and then sedimented in a sucrose gradient. In the control (Fig. 7 A) poly-[14ClU binds to ribosomes forming complexes of ribosomes-poly-[14CJU, which sediment in the region of lOO-2OO S (ref. 16). A similar sedimentation pattern was obtained in the presence of edeine (Fig. 7 B) thus indicating that edeine has no effect on the binding of poly-U to ribosomes.

E//ect o/ edeine on the polymerization o/ ribosome-bound [14C]phenylalanyl s-RNA In order to find whether edeine has an effect on the process of polymerization of ribosome-bound [14Clphenylalanyl s-RNA the ternary complex of ribosomespoly-U-E14Clphenylalanyl s-RNA was prepared and isolated after the method of 1NAKAMOTOs. This complex was incubated in a reaction mixture with edeine, phenylalanyl s-RNA and a soluble enzyme fraction 9. Simultaneously a sample without edeine was incubated and used as a control. After 5 rain of incubation at 28 ° poly[14Clphenylalanine was precipitated with hot 5 ~o trichloroacetic acid. The radioactivity of each precipitate is shown in Table I I I . These data show that edeine has

TABLE

III

EFFECT OF EDEINE ON THE POLYMERIZATION OF RIBOSOME-BOUND E14C]PHENYLALANYL s - R N A o.25-ml s a m p l e s c o n t a i n e d I 2 . 5 # m o l e s Tris b u f f e r (pH 7.4), 4 0 . ° / ~ m o l e s NH4C1, 2.o/~moles MgC1v 0.25/~moles d i s o d i u m salt of G T P , o.I 7 m g p h e n y l a l a n y l s - R N A , 5o.o/*g p o l y m e r i z i n g e n z y m e (fraction of 50-63 % [NH~]2SO 4 precipitate) a n d o.12 m g of t h e c o m p l e x of r i b o s o m e s poly-U-E14C]phenylalanyl s - R N A w i t h 259 c o u n t s / m i n . S a m p l e s were i n c u b a t e d for 5 mill a t 28 ° w i t h o u t or w i t h edeine as i n d i c a t e d in a b o v e table. R a d i o a c t i v i t y of h o t 5 % trichloroacetic acid p r e c i p i t a t e s w a s m e a s u r e d as described in M e t h o d s .

Conch. of edeine (l~M)

o 0.50 5.00

[14C]Phenylalanine Bound to ribosomes (counts/rain)

Hot trichloroacetic acid precipitate (counts /min )

259 259 259

228 226 216

Polymerization of E14C]phenylalanine (%)

88.0 85.o 83.o

no effect on the polymerization of phenylalanine from phenylalanyl s-RNA which has been previously bound to the complex of ribosome-poly-U.

DISCUSSION

The data presented in this paper show that edeine can be considered as an inhibitor of protein synthesis in vitro. The experiments carried out with various templates show that edeine does not inhibit amino acid incorporation into protein whose synthesis is dictated b y E. coli m-RNA, already naturally attached to the Biochim. Biophys. Acta, 95 (1965) 578-589

5~

M. HIEROWSKI, Z. KURYL()-B()I{OWSK.\

ribosomes. About 35 ~o inhibition occurs when an E. coli m - R N A fraction i~ added in vitro. The per cent of inhibition is higher (60 o//o) if f2 phage RNA is used as the tomplate RNA. The last observation is especially interesting in view of the fact that edeine inhibits the replication of f2 phage in vivo 3when used in the same concentrations as for the experiments in vitro. If synthetic polynucleotides are used as templates in the cell-free system, the inhibition induced b y edeine is even higher. The experiments with various synthetic polynucleotides, homopolymers as well as copolymers, show that edeine strongly inhibits the incorporation of specific amino acids into their homopolyamino acids. A 95 % inhibition is observed with poly-A-dependent incorporation of E14Cllysine and poly-U-dependent incorporation of El~Clphenylalanine. A little less inhibition (90 o/~) is observed with poly-C-dependent incorporation of I14Clproline. I t can be concluded that the inhibitory action of edeine occurs during polymerization of phenylalanine from aminoacyl s-RNA. More specifically it appears that edeine interferes with the binding of aminoacyl s-RNA to ribosomes. In view of the observation that edeine affects the formation of a ribosomespoly-U-phenylalanyl s-RNA complex, studies on the binding of edeine to ribosomes were undertaken. These results will be presented in the subsequent paper. Recent studies revealed that at the concentration of edeine which prevents the synthesis in vivo of DNA, the synthesis in vitro of DNA is decreased by about 50 °/(~. At the same concentration of edeine the synthesis in vitro of RNA is unaffected 3, however this concentration of edeine is IO times higher than that required for the maximal inhibition of polyphenylalanine synthesis. Comparison of the studies in vitro and in vivo seems to show differences in edeine action. Previous results have suggested that in vitro edeine inhibits I)NA synthesis b y affecting the activity of the polymerizing system. I t is not known whether the observed inhibition in vivo of DNA synthesis is due to changes in DNA polymerase activity, to inhibition of formation of a complex between DNA and this enzyme 17, or to a change in the rate of its synthesis. Further study will be required before the effects in vivo and in vitro of edeine can be interpreted.

ACKNOWLEDGEMENTS

This research was supported b y National Science Foundation and in part by Research Grant CA-o36IO from the National Cancer Institute, Public Health Science. One of us (M.H.) is a postdoctoral research fellow of the Rockefeller Foundation. I t is a great pleasure to acknowledge the advice and encouragement received from Dr. F. LIPMANN and Dr. E. L. TATUM during the course of this work.

REFERENCES i 2 3 4 5 6 7

Z. KURYLO-BoRoWSKA, Biochim. Biophys. Mcta, 61 (1962) 897. Z. KORYLO-BOROWSKA, Biochim. Biophys. Acta, 87 (1964) 3o5 . Z. KURYLO-BOROWSKA, u n p u b l i s h e d . M. A. NIRENBERG AND J. H. MATTHAEI, PrOC. Natl. Acad. Sci. U.S., 47 (1961) 158o. E. G. VON EHRENSTEIN AND F. LIPMANN, Proc. Natl. Mcad. Sci. U.S., 47 (1961) 941T. ~AKAMOTO, p e r s o n a l c o m m u n i c a t i o n , 1964. F. CHAPEVILLE, F. LIPMANN, G. VON EHRI~NSTEIN, B. WEISBLUM, W. J. RAY, Jr. AND S. BENZER, Proc. Natl. Mcad. Sci. U.S., 48 (1962) lO86.

Biochim. Biophys. Acta, 95 (1965) 578 589

ON THE MODE OF ACTION OF E D E I N E 8 9 io ii 12 13 14 15 16 17 18

589

F. SINGER AND J. K. G o s s , J. Biol. Chem., 237 (1962) 182. CONWAV, p e r s o n a l c o m m u n i c a t i o n , 1964. KURYLO-BOROWSKA, Bull. Inst. Marine Med. Gdansh, io (1959) 151. H. KNIPPENBERGER, H. VELDSTRA AND L. BOSCH, Biochim. Biophys. Acta, 80 (I964) 526. KUCAN AND F. LIPMANN, J. Biol. Chem., 239 (1964) 516. ~AKAMOTO, T. CONWAY, J. ALLENDE, G. SPYRIDES AND V. LIPMANN, Cold Spring Harbor Syrup. Quant. Biol., 28 (1963) 227. T. CONWAY, Proc. Natl. Acad. Sei. U.S., 51 (1964) 1216. G. SPYRIDES, Proc. Natl. Acad. Sci. U.S., 51 (1964) 122o. G. SPYRIDES AND F. LIPMANI% Federation Proe., 23 (1964) 219. D. BILLEN, Bioehim. Biophys. Acta, 68 (1963) 342. R. S. GARDNER, A. J. WAHBA, C. BASILIO, R. S. MILLER, P. LENGYEL AND J. F. SPEYER, Proc. Natl. Acad. Sci. U.S., 48 (I962) 2087.

M. T. Z. P. Z. T.

Bioehim. Biophys. Aeta, 95 (1965) 578-589